Increased separation efficiency via controlled aggregation of magnetic nanoparticles

ABSTRACT

Compositions and methods are disclosed which enhance the microscopic observation and analysis of biological entities such as cells, bacteria and viruses by eliminating interfering magnetic clusters created by naturally occurring aggregators of colloidal magnetic particles. Additionally means for significantly enhancing the magnetic isolation of low antigen density target cells from biological samples are disclosed.

PRIORITY INFORMATION

[0001] The present application is a continuation of U.S. applicationSer. No. 09/351,515 filed on Jul. 12, 1999.

FIELD OF THE INVENTION

[0002] This invention relates to the fields of bioaffinity separationsand diagnostic testing of biological samples. More specifically, theinvention provides compositions and methods which, may be used inmagnetic separation assays and enrichment procedures for controllingendogenous magnetic particle aggregation factors which, if uncontrolled,would obscure visualization of isolated entities. Also provided aremethods for constructing and synthesizing reversible aggregation factorsand the resulting compositions which simultaneously enhance recovery ofrare biological substances while facilitating observation of substancesso isolated.

BACKGROUND OF THE INVENTION

[0003] Several publications are referenced in this application bynumerals in parentheses in order to more fully describe the state of theart to which this invention pertains. The disclosure of each of thesepublications is incorporated by reference herein.

[0004] Many laboratory and clinical procedures employ bio-specificaffinity reactions. Such reactions are commonly utilized in diagnostictesting of biological samples, or for the separation of a wide range oftarget substances, especially biological entities such as cell, viruses,proteins, nucleic acids and the like. Various methods are available foranalyzing or separating the above-mentioned target substances based uponcomplex formation between the substance of interest and anothersubstance to which the target substance specifically binds. Separationof complexes from unbound material may be accomplished gravitationally,e.g. by settling, or, alternatively, by centrifugation of finely dividedparticles or beads coupled to the target substance. If desired, suchparticles or beads may be made magnetic to facilitate the bound/freeseparation step. Magnetic particles are well known in the art, as istheir use in immune and other bio-specific affinity reactions. See, forexample, U.S. Pat. No. 4,554,088 and Immunoassays for ClinicalChemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston,Edinborough (1983). Generally, any material which facilitates magneticor gravitational separation may be employed for this purpose. However,in the past 20 years the superiority of magnetics for performing suchseparations has led to its use in many applications.

[0005] Magnetic particles generally fall into two broad categories. Thefirst category includes particles that are permanently magnetizable, orferromagnetic. The second category comprises particles that demonstratebulk magnetic behavior only when subjected to a magnetic field. Thelatter are referred to as magnetically responsive particles. Materialsdisplaying magnetically responsive behavior are sometimes described assuperparamagnetic. However, materials exhibiting bulk ferromagneticproperties, e.g., magnetic iron oxide, may be characterized assuperparamagnetic only when provided in crystals of about 30 nm or lessin diameter. Larger crystals of ferromagnetic materials, by contrast,retain permanent magnet characteristics after exposure to a magneticfield and tend to aggregate thereafter due to strong particle-particleinteractions. Magnetic particles can be classified as large (1.5 toabout 50 microns), small (0.7-1.5 microns), and colloidal ornanoparticles (<200 nm). The latter are also called ferrofluids orferrofluid-like and have many of the properties of classicalferrofluids. Liberti et al pp 777-790, E. Pelizzetti (ed) “FineParticles Science and Technology” Kluwer Acad. Publishers, Netherlands,1996.

[0006] Small magnetic particles are quite useful in analyses involvingbio-specific affinity reactions, as they are conveniently coated withbiofunctional polymers (e.g., proteins), provide very high surface areasand give reasonable reaction kinetics. Magnetic particles ranging from0.7-1.5 microns have been described in the patent literature, including,by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685;4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of theseparticles are disclosed to be useful solid supports for immunologicreagents.

[0007] In addition to the small magnetic particles mentioned above,there are a class of large magnetic particles ranging in size fromapproximately 1.5-50 microns, which also have superparamagneticbehavior. Typical of such materials are those invented by Ugelstad (U.S.Pat. No. 4,654,267) and manufactured by Dynal, (Oslo, Norway). TheUgelstad process involves the synthesis of polymer particles which arecaused to swell and magnetite crystals are embedded in the swelledparticles. Other materials in the same size range are prepared bysynthesizing the particle in the presence of dispersed magnetitecrystals. This results in the trapping of magnetite crystals in apolymer matrix, thus making the resultant materials magnetic. In bothcases, the resultant particles have superparamagnetic behavior, which ismanifested by the ability to disperse readily upon removal of themagnetic field. Unlike magnetic colloids or nanoparticles, thesematerials, as well as small magnetic particles, are readily separatedwith simple laboratory magnetics because of the mass of magneticmaterial per particle. Thus, separations are effected in gradients fromas low as a few hundred gauss/cm on up to about 1.5 kilogauss/cm.Colloidal magnetic particles, (below approximately 200 nm),on the otherhand, require substantially higher magnetic gradients because of theirdiffusion energy, small magnetic mass per particle and Stokes drag.

[0008] U.S. Pat. No. 4,795,698 to Owen et al. relates to polymer-coated,colloidal, superparamagnetic particles. Such particles are manufacturedby precipitation of a magnetic species in the presence of abiofunctional polymer. The structure of the resulting particles,referred to herein as single-shot particles, has been found to be amicro-agglomerate in which one or more ferromagnetic crystallites havinga diameter of 5-10 nm are embedded within a polymer body having adiameter on the order of 50 nm. The resulting particles exhibit anappreciable tendency to remain in aqueous suspension for observationperiods as long as several months. U.S. Pat. No. 4,452,773 to Moldaydescribes a material similar in properties to those described in Owen etal., which is produced by forming magnetite and other iron oxides fromFe⁺²/Fe⁺³ via base addition in the presence of very high concentrationsof dextran. Materials so produced have colloidal properties and haveproved to be very useful in cell separation. This technology has beencommercialized by Miltenyi Biotec, Bergisch Gladbach, Germany.

[0009] Another method for producing superparamagnetic colloidalparticles is described in U.S. Pat. No. 5,597,531. In contrast to theparticles described in the Owen et al. patent, these latter particlesare produced by directly coating a biofunctional polymer onto pre-formedsuperparamagnetic crystals which have been dispersed, e.g., by sonicenergy into quasi-stable crystalline clusters ranging in size from about25-120 nm. The resulting particles, referred to herein as direct coated(DC) particles, exhibit a significantly larger magnetic moment than Owenet al. or Molday nanoparticles of the same overall size and can beseparated effectively in magnetic gradients greater than about 6kGauss/cm.

[0010] Magnetic separation techniques are known wherein a magnetic fieldis applied to a fluid medium in order to separate ferromagnetic bodiesfrom the fluid medium. In contrast, the tendency of colloidalsuperparamagnetic particles to remain in suspension, in conjunction withtheir relatively weak magnetic responsiveness, requires the use ofhigh-gradient magnetic separation (HGMS) techniques in order to separatesuch particles from a fluid medium in which they are suspended. In HGMSsystems, the gradient of the magnetic field, i.e., the spatialderivative, exerts a greater influence upon the behavior of thesuspended particles than is exerted by the strength of the field at agiven point.

[0011] High gradient magnetic separation is useful for separating a widevariety of magnetically labeled biological materials, includingeukaryotic and prokaryotic cells, viruses, nucleic acids, proteins, andcarbohydrates. In methods known heretofore, biological material has beenseparable by HGMS, provided at least one characteristic determinant ispresent on the material, which is capable of being specificallyrecognized and bound to a receptor, such as an antibody, antibodyfragment, specific binding protein (e.g., protein A, streptavidin),lectin, and the like.

[0012] HGMS systems can be divided into two broad categories. One suchcategory includes magnetic separation systems which employ a magneticcircuit that is entirely situated externally to a separation chamber orvessel. Examples of such external separators (or open field gradientseparators) are described in U.S. Pat. No. 5,186,827. In several of theembodiments described in the '827 patent, the requisite magnetic fieldgradient is produced by positioning permanent magnets around theperiphery of a non-magnetic container such that the like poles of themagnets are in a field-opposing configuration. The extent of themagnetic field gradient within the test medium obtainable in such asystem is limited by the strength of the magnets and the separationdistance between the magnets. Hence, there exists a finite limit togradients that can be obtained with external gradient systems. In aco-pending application Ser. No. 60/098,021, means for maximizing radialgradients and methods for maximizing separation efficiency via novelvessel designs are disclosed.

[0013] Another type of HGMS separator utilizes a ferromagneticcollection structure that is disposed within the test medium in orderto: (1) intensify an applied magnetic field; and (2) produce a magneticfield gradient within the test medium. Previously disclosed internalHGMS systems comprise fine steel wool or gauze packed within a columnthat is situated adjacent to a magnet. The applied magnetic field isconcentrated in the vicinity of the steel wires so that suspendedmagnetic particles will be attracted toward, and adhere to, the surfacesof the wires. The gradient produced on such wires is inverselyproportional to the wire diameter whereas the magnetic “reach” decreaseswith diameter. Hence, very high gradients can be generated.

[0014] One major drawback of internal gradient systems is that the useof steel wool, gauze material, steel microbeads or the like, may entrapnon-magnetic components of the test medium by capillary action in thevicinity of intersecting wires or within interstices betweenintersecting wires. Various coating procedures have been applied to suchinternal gradient columns (U.S. Pat. Nos. 5,693,539; 4,375,407),however, the large surface area in such systems still creates recoveryproblems due to absorption. Hence, internal gradient systems are notdesirable, particularly when recovery of very low frequency capturedentities is the goal of the separation. Further, these systems makeautomation difficult and costly.

[0015] On the other hand, HGMS approaches using external gradients forcell separation provide a number of conveniences. Firstly, simplelaboratory tubes such as test tubes, centrifuge tubes or evenvacutainers (used for blood collection) can be employed. When externalgradients are of the kind in which separated cells are effectivelymonolayered, as is the case with quadrupole/hexapole devices (U.S. Pat.No. 5,186,827) or the opposing dipole arrangement described in U.S. Pat.No. 5,466,574, washing of cells or subsequent manipulations arefacilitated. Further, recoveries of cells from tubes or similarcontainers is a simple and efficient process. This is particularly thecase when compared to recoveries from high gradient columns. Suchseparation vessels also provide another important feature which is theability to reduce volume of the original sample. For example, if aparticular human blood cell subset, (e.g. magnetically labeled CD 34+cells), is isolated from blood diluted 20% with buffer to reduceviscosity, a 15 ml conical test tube may be employed as the separationvessel in an appropriate quadrupole magnetic device. After appropriatewashes and/or separations and resuspensions to remove non-bound cells,CD34+ cells can very effectively be resuspended in a volume of 200 μl.

[0016] This can be accomplished, for example, by starting with 12 ml ofsolution (blood, ferrofluid and dilution buffer) in a 15 ml conical testtube, performing a separation, discarding the “supernatant” andsubsequent wash “supernatants” and resuspending the recovered cells in 3ml of appropriate cell buffer. A second separation is then performedwhich may include additional separation/wash steps (as might benecessary for doing labeling/staining reactions) and finally theisolated cells are easily resuspended in a final volume of 200 μl. Byreducing volume in this sequential fashion, and employing a vortex mixerfor resuspension, cells adhered to the tube above the resuspensionvolume are recovered into the reduced volume. When done carefully andrapidly in appropriately treated vessels, cell recovery is quiteefficient, ranging between 70-90%.

[0017] The efficiency with which magnetic separations can be done andthe recovery and purity of magnetically labeled cells will depend onmany factors. These include such considerations as the number of cellsbeing separated, the receptor density of such cells, the magnetic loadper cell, the non-specific binding (NSB) of the magnetic material, thetechnique employed, the nature of the vessel, the nature of the vesselsurface, the viscosity of the medium and the magnetic separation deviceemployed. If the level of non-specific binding of a system issubstantially constant, as is usually the case, then as the targetpopulation decreases so does the purity. As an example, a system with0.2% NSB that recovers 80% of a population which is at 0.25% in theoriginal mixture will have a purity of 50%. Whereas if the initialpopulation were at 1.0%, the purity would be 80%. Not as obvious is thefact that the smaller the population of a targeted cell, the moredifficult it will be to magnetically label and to recover. Furthermore,labeling and recovery will markedly depend on the nature of magneticparticle employed. For example, when cells are incubated with largemagnetic particles, such as Dynal beads, the cells are labeled throughcollisions created by mixing of the system as the beads tend to be toolarge to diffuse.

[0018] Thus, if a cell were present in a population at a frequency of 1cell/ml of blood or even less, as could be the case for tumor cells invery early cancers, then the probability of labeling target cells willbe related to the numbers of magnetic particles added to the system andthe length of time of mixing. Since mixing of cells with such particlesfor substantial periods of time will be deleterious, it becomesnecessary to increase particle concentration as much a possible. Thereis, however, a limit to the quantity of magnetic particle that can beadded to the system, in that one can substitute a system comprising arare cell mixed in with other blood cells with one comprising a rarecell mixed in with large quantities of magnetic particles uponseparation, in which case the ability to enumerate the cells of interestor to examine them is not markedly improved.

[0019] There is another drawback to the use of large particles toisolate cells having rare frequencies (1-50 cells/ml of blood). Despitethe fact that large magnetic particles allow the use of externalgradients of very simple design and relatively low magnetic gradient,large particles tend to cluster around cells in a cage-like fashionmaking them difficult to “see” or to analyze. Hence, the particles mustbe released before analysis, and releasing the particles oftenintroduces other complications.

[0020] In theory, colloidal magnetic particles, used in conjunction withhigh gradient magnetic separation, should be the method of choice forseparating a cell subset of interest from a mixed population ofeukaryotic cells, particularly if the subset of interest comprises onlya small fraction of the entire population. With appropriate magneticloading, sufficient force is exerted on a cell, facilitating itsisolation even in a media as viscous as moderately diluted whole blood.As noted, colloidal magnetic materials below about 200 nanometers willexhibit Brownian motion which markedly enhances their ability to collidewith and magnetically label rare cells. This is demonstrated in U.S.Pat. No. 5,541,072, where results of very efficient tumor cell purgingexperiments are described employing 100 nm colloidal magnetic particles(ferrofluids). Just as importantly, colloidal materials at or below thesize range noted do not generally interfere with viewing of cells. Cellsso retrieved can be examined by flow cytometry with minimal forwardscattering effects or by microscopy employing visible or fluorescenttechniques. Because of their diffusive properties, such materials, incontrast to large magnetic particles, readily “find” and magneticallylabel rare biological entities such as tumor cells in blood.

[0021] There is, however, a significant problem which arises in the useof ferrofluid-like materials for cell separation in external fieldgradient systems which, for reasons given above, is the device design ofchoice. Direct monoclonal antibody conjugates of Owen et al. materialsor Molday nanoparticles, such as those produced by Miltenyi Biotec, donot have sufficient magnetic moment for use in cell selection employingthe best available external magnetic gradient devices, such as thequadrupole or hexapole magnetic devices described in U.S. Pat. No.5,186,827. When used for separations in moderately diluted whole blood,they are even less effective. Using similar materials, which aresubstantially more magnetic, as described in U.S. Pat. No. 5,698,271,more promising results have been obtained. In model spiking experiments,it has been found that SKBR3 cells (breast tumor line), which have ahigh EpCAM (epithelial cell-adhesion molecule) determinant density, areefficiently separated from whole blood with direct conjugates of antiEpCAM MAb ferrofluids even at very low spiking densities (1-5 cells/mlblood). On the other hand, PC3 cells (a prostate tumor line) which havelow antigen density are separated at significantly lower efficiency.Most likely this is a consequence of inadequate magnetic loading ontothese low density receptor cells.

[0022] From the foregoing discussion, it would be advantageous toprovide a magnetic separation system which combines the beneficialproperties of both colloidal magnetic materials and large magneticparticles (e.g., diffusion based labeling and large magnetic moment,respectively) for separations involving rare events or for cells withvery low density receptors. One could envision starting a separationprocess with a magnetic colloidal or nanoparticle which, due to theirBrownian motion, would rapidly find and label cells in rare numbers orcells with very low density receptors. Once that labeling is achieved,it would be desirable to convert the magnetic moment of the nanoparticleto a value similar to that of a large magnetic particle. In that way,magnetically labeled entities could be separated in the kinds ofgradient fields used for larger particles, e.g., a simple external fieldgradient separator. In the case of very low density receptor cells,which are recovered inefficiently even in high gradient external fieldseparators, use of such a principle would clearly increase theefficiency of separation. In applications where cells are to be analyzedor used for some biological purpose following separation, it would alsobe very desirable to be able to convert the magnetic moment of thelabeled entity back to that of its original colloidal magnetic labelingdensity. This approach would permit separation from excessive magneticmaterial, which would facilitate subsequent analysis or use.

[0023] U.S. Pat. No. 5,466,574 to Liberti et al., describes a systemwhich has some of the foregoing features regarding “loading on” ofmagnetic materials onto cells. It was discovered that when cells werefirst labeled with specific monoclonal antibodies (with or withoutbiotinylation) followed by magnetic labeling with goat anti-mouseferrofluid or with streptavidin-ferrofluid (respectively), separationwas enhanced in the presence of excess monoclonal antibody. The uniqueability of ferrofluids to create this “no wash” enhancing procedure isdue to immunochemical crosslinking of free ferrofluid in solution toferrofluid-bound target cells. Ferrofluid bound to monoclonal antibodyon cells, in turn, binds to free ferrofluid in solution via freemonoclonal antibody. This results in immunochemical clusters ofmonoclonal antibody/ferrofluid “growing” off of monoclonal antibodylabeled cell determinants (referred to as chaining). Thus, magneticcolloid is “artificially” loaded onto cells making them more magneticand easier to separate. The phenomenon was found to obey immunochemicalrules, in that a high excess of monoclonal antibody resulted in adecrease in chaining (monoclonal excess zone) and a loss of separationefficiency. Similarly high levels of ferrofluid also reduced chaining(ferrofluid excess zone). Chaining has been found to be useful forpurging unwanted cells, e.g. tumor cells, in bone marrow or peripheralblood “grafts.” By this method, very high levels of magnetic material(visible brown rims around cells, as observed via microscopy) can beloaded onto target cells giving rise to very efficient separation inhigh gradient fields of only 8-12 kGauss/cm gradients. On the otherhand, cells labeled with “monomeric” ferrofluid were found to separateless efficiently in the same gradient.

[0024] In attempts to use chaining for isolating rare cells from wholeblood, several problems have been encountered. First, although spikedcells are, indeed, efficiently recovered, they are so densely coveredwith ferrofluid (chaining) that the ability to analyze them is markedlyreduced. Hence this approach is not ideal for applications wherein thepositively selected cells are to be observed via microscopy or flowcytometry. Additionally, chaining seems to promote non-specific binding.In summary, designing a chaining-based assay where the level of chainingsimultaneously gives rise to separation enhancement, non-obstructedviewing of the isolated cells and acceptable levels of non-specificbinding is extraordinarily difficult. The chaining reaction is difficultto control because it requires immunochemical stoichiometry. Forexample, most (>99%) of the added monoclonal antibody (or taggingligand) will always be free in solution regardless of the affinity ofthe antibodies. Hence, the amount of ferrofluid required to achieveimmunochemical equivalence (where the best separations take place viachaining) generally leads to more chaining than is desired, particularlyin the case where the selected cell is to be viewed and/or furtherstudied. Chaining can be lessened by concurrent decreases in labelingmonoclonal antibody and added ferrofluid, however this results in asacrifice of separation efficiency. Another drawback to the use ofchaining to enhance separation is the inability to, in some practicalmanner, reverse chaining. If chaining could be reversed and theconcomitant increase in non-specific binding decreased, the phenomenonwould provide a viable approach to enabling the desired “loading on” ofmagnetic material. Another disadvantage of this method is that a twostep reaction is required, i.e., reaction of targets with primarymonoclonal antibody in a first step followed by repetition withferrofluid specific for primary monoclonal antibody in the second step.This approach cannot be used in assays where primary antibody isdirectly conjugated to ferrofluid.

[0025] U.S. Pat. No. 5,108,933 to Liberti et al. discloses the use ofweakly magnetic colloidal materials such as those described by Owen etal. or Molday in immunoassays employing external field magneticseparators. Such materials are described therein as agglomerable andresuspendable colloidal magnetic materials which remain substantiallyundisturbed in an external magnetic field system, for example, thosecommercially available at that time (Ciba Corning, Wampole, Mass.;Serono Diagnostics, Norwell, Mass.). By contrast, materials made by theprocess disclosed in the '531 patent being substantially more magnetic,as noted above, will separate in those separators. In the '933 patentmeans for converting the colloid to an agglomerate are disclosed so asto make them separable in those separators. Thus, such materials couldbe used for performing the bound/free separation step of immunoassays.There is no mention in '933 for the need of, or methods for reversingagglomeration reactions.

[0026] In light of the foregoing and recent discoveries of naturallyoccurring ferrofluid aggregation factors, the present inventors haverecognized the need for compositions and methods for controllingaggregation of ferrofluid by endogenous factors during the isolation andimmunochemical characterization of rare target bioentities. Suchcompositions and methods may be used to advantage to facilitate analysisand observation of bioentities so isolated. Further, this invention alsopermits the use of substantially less magnetic reagent as well as theopportunity to use lower magnetic gradients. In the case of a fixedgradient, the invention provides for the capture or isolation ofentities which might have otherwise had insufficient magnetic labelingto be captured.

SUMMARY OF THE INVENTION

[0027] In accordance with the present invention, methods, compositionsand kits are provided for controlling the aggregation of ferromagneticnanoparticles by endogenous aggregation factors. Ferrofluid aggregationoften presents problems during subsequent viewing of the isolatedtargets. The methods of the invention facilitate visualization of theisolated bioentities by allowing the investigator to control the levelof aggregation. In one embodiment of the invention, a method is providedfor inhibiting the aggregation of magnetic nanoparticles on the surfaceof isolated target entities. The method comprises obtaining a biologicalspecimen suspected of containing a target bioentity. Next,immunomagnetic suspensions are prepared by mixing the specimen withcolloidal, magnetic particles coupled to a biospecific ligand havingaffinity for at least one characteristic determinant of the targetbioentity. The immunomagnetic suspension is thereafter subjected to amagnetic field to obtain target bioentity enriched fractions.optionally, the fractions are then examined to determine thecharacteristics of the target bioentity so isolated. Inhibition offerrofluid aggregation facilitates subsequent analysis of cells asaggregates of ferrofluid on the cell surface are eliminated. The absenceof such aggregates is important for several types of analyses including,for example, flow cytometry and immunofluorescence microscopy.

[0028] The reagents provided herein efficiently inhibit or removeendogenous aggregation factors. The factor removal or inhibition stepmay be performed before or simultaneously with the addition offerrofluid to the biological specimen for separation and enrichment.

[0029] To further characterize target bioentities isolated using themethods of the invention, the method optionally includes the steps ofadding to the target bioentity enriched fraction at least onebiospecific reagent which recognizes and effectively labels at least oneadditional characteristic determinant on said target bioentity. Thelabeled target bioentities are then separated in a magnetic field toremove unbound biospecific reagents. A non-cell exclusion agent is addedto the separated bioentities to allow exclusion of non-nucleatedcomponents present in the sample. After purifying the target bioentity,it is then ready for analysis using a variety of different analysisplatforms. Target bioentities include, without limitation, tumor cells,virally infected cells, fetal cells in maternal circulation, virusparticles, bacterial cells, white blood cells, myocardial cells,epithelial cells, endothelial cells, proteins, hormones, DNA, and RNA.Target bioentities may be analyzed by a process selected from the groupconsisting of multiparameter flow cytometry, immunoflourescentmicroscopy, laser scanning cytometry, bright field base image analysis,capillary volumetry, manual cell analysis and automated cell analysis.Aggregation inhibiting agents suitable for use in the methods of thepresent invention, include, but are not limited to reducing agents,animal serum proteins, immune-complexes, carbohydrates, chelating agent,unconjugated ferrofluid, and diamino butane. In the case where theendogenous aggregation factor is of the IgM class and reactive withferrofluids, preferred aggregation inhibiting agents are reducingagents, such as Mercapto ethane sulfonic acid [MES], Mercapto PropaneSulfonic acid [MPS] and dithiothreitol [DTT]. In a particularlypreferred embodiment, the biospecific ligand is a monoclonal antibodyhaving affinity for an epithelial cell adhesion molecule.

[0030] In an alternative and preferred embodiment of the invention, amethod is provided for isolating target bioentities from a biologicalsample by controlling aggregation of magnetic nanoparticles. The methodentails obtaining a biological specimen suspected of containing saidtarget bioentity and contacting the biological specimen with a reagenteffective to inactivate any endogenous aggregating factors present.Immunomagnetic suspensions are then prepared wherein the specimen ismixed with colloidal, magnetic particles coupled to a biospecific ligandhaving affinity for at least one antigen present on the targetbioentity, the magnetic particles being further coupled to a firstexogenous aggregation enhancing factor which comprises a first member ofa specific binding pair. A second multivalent exogenous aggregationenhancing factor is then added to the immunomagnetic suspension toincrease aggregation of the particles, the second aggregating enhancingfactor comprising the second member of the specific binding pair, whichreversibly binds to the magnetically labeled target bioentity. Thesample is then subjected to a magnetic field to obtain a targetbioentity enriched fraction. This preferred embodiment takes advantageof the fact that aggregating ferrofluid onto target entities in acontrolled and reversible fashion results in substantially improvedisolation efficiency.

[0031] In a further embodiment, the above described method furthercomprises the steps of adding at least one biospecific reagent whichrecognizes and labels at least one additional characteristic determinanton said target bioentity. The target bioentity so labeled is thenseparated in a magnetic field to remove unbound biospecific reagent. Anon-cell exclusion agent is added to the separated bioentities to allowexclusion of non-nucleated components present in the sample. The targetbioentity is then purified and further analyzed. In order to reverse theaggregation mediated by the exogenous aggregation factors, a member ofthe specific binding pair may be added in excess to the purifiedbioentity to reduce ferrofluid aggregation on the surface of cells,thereby facilitating viewing of the cells, e.g. in a microscope.Suitable specific binding pairs for this purpose include, withoutlimitation, biotin-streptavidin, antigen-antibody, receptor-hormone,receptor-ligand, agonist-antagonist, lectin-carbohydrate, ProteinA-antibody Fc, avidin-biotin, biotin analog-streptavidin, biotinanalog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin,iminobiotin-streptavidin, and iminobiotin-avidin. Preferably, thebiospecific ligand is a monoclonal antibody having affinity forepithelial cell adhesion molecule. Exemplary biospecific reagentsinclude monoclonal antibodies, polyclonal antibodies, detectably labeledantibodies, antibody fragments, and single chain antibodies. Isolatedtarget bioentities may be analyzed by a process selected from the groupconsisting of multiparameter flow cytometry, immunofluorescentmicroscopy, laser scanning cytometry, bright field base image analysis,capillary volumetry, manual cell analysis and automated cell analysis.

[0032] In accordance with the present invention, controlling aggregationof ferrofluid in a sample has several unexpected benefits previouslynoted, e.g. increasing efficiency of separation of some particularentity. It has been discovered that addition of an exogenous aggregationenhancing factor gives rise to increased magnetic loading, resulting inincreased separation efficiency while reducing the amount of ferrofluidrequired to isolate the target bioentity. The increased magnetic loadingalso allows for reduced incubation periods and facilitates isolation ofthe target bioentity in the presence of a suboptimal magnetic field.

[0033] In an additional embodiment of the present invention a kit isprovided which facilitates the practice of the methods described herein.An exemplary kit for isolating target bioentities includes i) coatedmagnetic nanoparticles comprising a magnetic core material, a proteinbase coating material, and an antibody that binds specifically to afirst characteristic determinant of said target bioentity, said antibodybeing coupled, directly or indirectly, to said base coating material;ii) at least one antibody having binding specificity for a secondcharacteristic determinant of said rare biological substance; iii) anaggregation inhibiting factor; and iv) a non-cell exclusion agent forexcluding non-nucleated sample components other than said targetbioentity from analysis.

[0034] A kit for improving the isolation efficiency of certainbiological entities, such as might be required for isolating low antigendensity tumor cells from a biological sample, is also provided inaccordance with the present invention. This kit utilizes controlled andreversible aggregation of magnetic nanoparticles to achieve suchimprovement. Such a kit includes i) a reagent effective to inactivateendogenous aggregating factors; ii) coated magnetic nanoparticlescomprising a magnetic core material, a protein base coating material,and an antibody that binds specifically to a first characteristicdeterminant of said tumor cell, the antibody being coupled, directly orindirectly, to the base coating material; the magnetic particles beingfurther coupled to a first exogenous aggregation enhancing factor, thefactor comprising one member of a specific binding pair; iii) at leastone antibody having binding specificity for a second characteristicdeterminant of said tumor cell; iv) a second exogenous aggregationenhancing factor, the second aggregation enhancing factor comprising thesecond member of the specific binding pair; and v) a non-cell exclusionagent for excluding non-nucleated sample components other than the tumorcells from analysis. The kit may optionally include a reagent forreversing the exogenous aggregation factor. Specific binding pairsuseful in such a kit, include without limitation, biotin-streptavidin,antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist,lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotinanalog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin,iminobiotin-streptavidin, and iminobiotin-avidin. Reagents effective toinactivate endogenous aggregating factors include reducing agents,animal serum proteins, immune-complexes, carbohydrates, chelating agent,unconjugated ferrofluid, and diamino butane.

[0035] The methods, compositions and kits of the invention provide themeans for controlling the aggregation of magnetic nanoparticles, thusfacilitating the isolation, visualization and characterization of rarebiological substances or cells from biological specimens.

BRIEF DESCRIPTION OF THE DRAWING

[0036] FIGS. 1A-1H are a series of micrographs depicting what isobserved in a microscope in samples derived from blood donors with highlevels of endogenous aggregating factors versus those with low levels ofendogenous aggregation factors. Breast cancer cells were spiked intowhole blood and selected using EPCAM colloidal magnetic particles andstained in suspension. FIG. 1A, transmitted light only, low aggregation;FIG. 1B, transmitted light only, high aggregation; FIG. 1C, cellsstained with Hoechst nuclear stain, low aggregation; FIG. 1D, cellsstained with Hoechst nuclear stain, high aggregation; FIG. 1E, cellsstained with the epithelial cell marker cytokeratin Alexa 488, lowaggregation; FIG. 1F, cells stained with the epithelial cell markercytokeratin Alexa 488, high aggregation; FIG. 1G, cells stained withtumor cell receptor marker erb2-conjugated to phycoerythrin, lowaggregation; FIG. 1H, cells stained with tumor cell receptor markererb2-conjugated to phycoerythrin, high aggregation.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The term “target bioentities” as used herein refers to a widevariety of materials of biological or medical interest. Examples includehormones, proteins, peptides, lectins, oligonucleotides, drugs, chemicalsubstances, nucleic acid molecules, (e.g., RNA and/or DNA) andparticulate analytes of biological origin, which include bioparticlessuch as cells, viruses, bacteria and the like. In a preferred embodimentof the invention, rare cells, such as fetal cells in maternalcirculation, or circulating cancer cells may be efficiently isolatedfrom non-target cells and/or other bioentities, using the compositions,methods and kits of the present invention. The term “biologicalspecimen” includes, without limitation, cell-containing bodily, fluids,peripheral blood, tissue homogenates, nipple aspirates, and any othersource of rare cells that is obtainable from a human subject. Anexemplary tissue homogenate may be obtained from the sentinel node in abreast cancer patient. The term “determinant”, when used in reference toany of the foregoing target bioentities, may be specifically bound by abiospecific ligand or a biospecific reagent, and refers to that portionof the target bioentity involved in, and responsible for, selectivebinding to a specific binding substance, the presence of which isrequired for selective binding to occur. In fundamental terms,determinants are molecular contact regions on target bioentities thatare recognized by receptors in specific binding pair reactions. The term“specific binding pair” as used herein includes antigen-antibody,receptor-hormone, receptor-ligand, agonist-antagonist,lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fcreceptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin andvirus-receptor interactions. Various other determinant-specific bindingsubstance combinations are contemplated for use in practicing themethods of this invention, such as will be apparent to those skilled inthe art. The term “antibody” as used herein, includes immunoglobulins,monoclonal or polyclonal antibodies, immunoreactive immunoglobulinfragments, and single chain antibodies. Also contemplated for use in theinvention are peptides, oligonucleotides or a combination thereof whichspecifically recognize determinants with specificity similar totraditionally generated antibodies. The term “detectably label” is usedto herein to refer to any substance whose detection or measurement,either directly or indirectly, by physical or chemical means, isindicative of the presence of the target bioentity in the test sample.Representative examples of useful detectable labels, include, but arenot limited to the following: molecules or ions directly or indirectlydetectable based on light absorbance, fluorescence, reflectance, lightscatter, phosphorescence, or luminescence properties; molecules or ionsdetectable by their radioactive properties; molecules or ions detectableby their nuclear magnetic resonance or paramagnetic properties. Includedamong the group of molecules indirectly detectable based on lightabsorbance or fluorescence, for example, are various enzymes which causeappropriate substrates to convert, e.g., from non-light absorbing tolight absorbing molecules, or from non-fluorescent to fluorescentmolecules. A nucleic acid dye or other reporter molecule, sometimesreferred to herein as a non-cell exclusion agent, which is capable ofidentifying both target bioentities and certain non-target bioentities,such as intact nucleated cells, is added to the sample to allowexclusion of any residual non-nucleated cells or other potentiallyinterfering sample components prior to analysis by flowcytometry,microscopy, or other analytical platforms. Non-cell exclusion agents maybe reactive with DNA, RNA, protein, or lipids such that the amount ofsignal obtained is typical of that obtained for cells or the imageobtained reveals typical features of a cell, such as cell and nuclearmembranes, nucleus, and mitochondria.

[0038] The term “optimal” used herein describing ferrofluidconcentration, magnetic field strength, or incubation time refers to theconditions used in a standard, unmodified assay, separation, isolation,or enrichment.

[0039] The term “sub-optimal” used herein describing ferrofluidconcentration, magnetic field strength, or incubation time refers to theconditions used in an assay, separation, isolation, or enrichment whichwould produce inferior results, as compared to results obtainable underoptimal conditions.

[0040] The term “ferrofluid” as used herein refers to magneticnanoparticles in suspension. The terms ferrofluid and magneticnanoparticles are used interchangeably herein.

[0041] Endogenous ferrofluid aggregation factors are those present in asample isolated from a test subject. Exogenous aggregation factors arethose provided herein which are added and/or reversed as desired by theinvestigator.

[0042] The preferred magnetic particles for use in this invention areparticles that behave as colloids and are superparamagnetic. Thecolloids are characterized by their size, i.e., smaller than 200 nm orof a size which doesn't interfere with the analysis. Thesuperparamagnetic particles become magnetic only when they are subjectedto a magnetic field gradient and do not become permanently magnetic. Thecolloidal superparamagnetic particles do not separate or settle fromaqueous solution for extended periods of time. These particles arecomposed of either single crystals of iron oxides or agglomerates ofsuch crystals surrounded by molecules either physically adsorbed orcovalently attached. The colloidal magnetic particles with the abovecharacteristics can be prepared as described in U.S. Pat. Nos.4,795,698; 5,512,332 and 5,597,531. A monoclonal antibody, whichrecognizes a specific subset of cells, conjugated to magnetic particlesis preferred for use in this invention.

[0043] In the course of studies on rare cell isolation, a factor presentin the blood of certain patients was discovered which effectuatesaggregation of magnetic nanoparticles. Further, this enhancedaggregation effect appears to be readily reversible. Thus, rare cellshaving low receptor densities can be isolated more efficiently frommoderately diluted whole blood in external field quadrupole or hexapoleseparators by “loading on” more ferrofluid than that which isspecifically bound to a characteristic determinant of the rare cell ofinterest. When isolated cells are examined by microscopy, ferrofluidclusters are present on the cell membrane. Reversal of the enhancingeffect deaggregates the clusters, facilitating microscopic analysis ofthe cells. This endogenous enhancing effect exists in varying levels inabout 90% of apparently normal donor blood samples. By manipulatingassay conditions, the resultant ferrofluid clusters can be dispersedwithout damaging the cells of interest. Thus, the present inventionprovides methods for eliminating the effects of endogenous ferrofluidaggregation factors and where desirable, means for constructing agentscapable of controlled and reversible aggregation of ferrofluid. Thispermits effective and efficient isolation and enrichment and subsequentanalysis of rare cells, and other biological entities such as virusesand bacteria.

[0044] The endogenous ferrofluid aggregating substance found in bloodhas the following characteristics: (1) it is present in plasma or serum;(2) it is sensitive to millimolar concentrations of dithiothreitol; and(3) it reacts with “bare” crystalline regions on direct coatedferrofluid, as described in U.S. Pat. No. 5,597,531. Control experimentsrevealed that the factor is not ferritin, transferrin, fibrinogen, C1q,human anti-mouse or anti-BSA antibodies. The aggregating substance isalso not of the IgG subclass since IgG-depleted plasma also causedaggregation of the ferrofluid. Ferrofluid aggregation appears to becorrelatable with ferrofluid or serum concentration. The dependency ofaggregation on concentration of either component is similar to thatobserved with precipitin curves and based on the observations notedabove, it was postulated that the substance is an IgM. This wasconclusively proven by removing the IgM from the plasma samples viaimmunoaffinity purification. The resulting IgM depleted plasma did noteffectuate ferrofluid aggregation.

[0045] Control experiments, where the same plasma samples were absorbedthrough a BSA affinity media, did show ferrofluid aggregation. Furtherstudy demonstrated that extensive adsorption of sera containing theaggregating factor with ferrofluids only removed a small amount of thetotal IgM and that purified human IgM causes ferrofluid aggregation.These observations along with identity studies of adsorbed aggregatingfactor and the ability to cause aggregation with adsorbed/desorbedmaterial led to the conclusion that the aggregation factor in theseexperiments is, indeed, a highly specific IgM. Based on the inability toinhibit aggregation by any component used in forming the ferrofluidexcept for magnetite crystals poorly coated with protein or magnetitecrystals partially coated with detergent, it is believed that theepitope recognized by the IgM is present on the magnetite crystallinesurface. The role of this specific IgM is not known but it is present ina significant portion of the human population and at varying levels. Itis possible that this antibody plays some role in iron metabolism.

[0046] Methods of manipulating endogenous enhancing factors aredescribed herein which permit patient-to-patient comparison of isolatedcells in a meaningful manner. Because the endogenous aggregationenhancing factor concentration varies in patient populations, it isdifficult to create a standard procedure for separating rare circulatingcells, be they tumor cells, fetal cells in maternal blood or virallyinfected cells. Recent discoveries in a co-pending application Ser. No.09/248,388) have demonstrated that the magnitude of the number ofcirculating cells is directly related to tumor burden and stage ofdisease in breast cancer patients. Similarly, viral burden has beenshown to be of significance in the prognosis of HIV infection. The needfor accurate quantitation of such entities is becoming ever moreimportant. The methods disclosed herein are conveniently practiced bymeans of test kits which may be used to advantage in the clinicalsetting.

[0047] Having discovered the presence of this ferrofluid aggregationfactor (FFAF) in donor sera, careful studies confirmed that its presencemarkedly improves the retrieval of spiked low density receptor tumorcells using monoclonal antibody conjugated ferrofluid. In these studies,cells spiked at levels of 1 cell/2 mls of blood containing FFAF areroutinely retrieved at greater than 70% efficiency. In contrast, indonor blood where FFAF is absent, efficiency of retrieval is reduced toapproximately 15-25%. From those experiments, the following observationswere made:

[0048] 1. low density receptor cells are often isolated less efficientlythan cells with high density receptors;

[0049] 2. separation efficiency varies considerably for low densityreceptor cells and this variation is dependent on the blood donor;

[0050] 3. when either cell type (high or low density characteristicdeterminant cells) was examined via microscopy followingferrofluid/magnetic isolation from the blood of different donors,ferrofluid was aggregated in about 90% of patient samples; and

[0051] 4. tumor cells retrieved had differing degrees of visibleferrofluid aggregated on their surfaces.

[0052] Moreover, since the aggregation material is sensitive to DTT,retrieved cells can be readily visualized by microscopy formorphological characteristics by reduction of the ferrofluid aggregatesprior to observation.

[0053] In comparing the phenomena of FFAF and chaining (see U.S. Pat.No. 5,466,574) to enhance cell recoveries, some interesting conclusionscan be drawn. When FFAF is present in blood in exceedingly lowconcentrations, as is the case in many individuals, excellent recoveriesof low density receptor spiked cells are obtained, e.g., 1-5 cells/mlblood. The quality of the recovered cells, as assessed by the level ofmagnetic material on their surfaces is quite suitable for morphologicalexamination or further manipulation. When FFAF is in highconcentrations, recoveries are also excellent but the quality of therecovered cells is not acceptable as the agglomerates on the cellsobscures their viewing. Hence, unlike the components of chaining, i.e.monoclonal antibody-ferrofluid “chains”, which are limited byconcentration, there is a level of FFAF which can lead to very effectivecell separation, thus providing an isolated population of cells whichcan be studied effectively. In certain embodiments of the invention,endogenous FFAF's in the samples are inactivated at the outset so thataggregation can be controlled by the investigator by adding thecompositions described herein. In this way, aggregation can be enhancedor inhibited as desired.

[0054] FFAF and any similar substances must be evaluated in assays toisolate rare cells. Such factors must be controlled in order to developa test which functions reliably for every patient. For example, manyindividuals have anti-rodent antibodies, or antibodies to components,such as carbohydrates, which might be found on the surface of somemagnetic nanoparticles. Other potential aggregating substances normallypresent in blood include C1q, rheumatoid factor, and blood clottingproteins. Such reactions must either be controlled, so as to make themconstant from specimen to specimen, or be eliminated altogether.

[0055] In the case of anti-rodent antibodies, this can be accomplishedby adding rodent proteins to the system so as to inhibit theiraggregating effects. This addition is quite different from adding suchcomponents to immunoassay systems. In the latter case, anti-rodentantibodies generally enhance isolation but also tend to increase falsepositives as they link captured antibody with labeling antibody insandwich-type reactions. In contrast, additional antibodies used in themethods of the present invention enhance the recovery of low densityreceptor cells. Thus, in one case aggregating factors can artificiallyelevate false positives, and in another facilitate isolation of thetarget bioentity.

[0056] Besides competitively inhibiting such factors, they may bedisabled or adsorbed from solution, or the determinant on the magneticcolloid to which such factors bind may be eliminated. FFAF activity maybe inhibited by formulating a special buffer that contains optimalamounts of additives such as mouse and other animal serum proteins,immune-complexes, carbohydrates, chelating agents which inhibit variousactivation systems, including complement, and compounds whichspecifically inhibit the interaction of C1q with reacted antibody, suchas diamino butane. In the case where FFAF is an IgM, reducing agentseffectively disable the FFAF without affecting the ligands used forlabeling cells. Thus, such factors could be selectively disabledchemically or enzymatically. Adsorption with appropriate materials,e.g., unconjugated ferrofluid, provides another route for removingaggregating factors from the sample.

[0057] It may also be possible to make the amount of aggregator constantfrom patient to patient so as to always have identical levels ofenhancement. This may be accomplished in different ways. For example,all endogenous factors may be reduced to the same level in all patientsamples. However this seems a difficult and impractical solution. As analternative, a two step process may be employed where, as a first step,all endogenous FFAF's are disabled without affecting specific ligandbinding of the ferrofluid to its target or subsequent cell analysis. Thesecond step entails a controlled aggregation reaction. Colloidalmagnetic materials conjugated to two kinds of ligands may be used topractice the two step method described above. One ligand, such asmonoclonal antibody would be directed to cell surface determinants andeffectively labels cells. The secondary ligand would have no reactivitywith any component of blood yet have binding affinity for a multivalentcomponent which would be added following binding of the primary ligand.Thus, additional magnetic colloid would bind to colloid already bound tocells, thereby enhancing their magnetic load just as FFAF does.Similarly, it would be preferable if the reaction of the secondaryligand and its multivalent component were reversible. By selecting theright components of the secondary reactions and their concentrations, itshould be possible to add ferrofluid and the aggregation factorsimultaneously.

[0058] Therefore, FFAFs are provided which facilitate controlledaggregation of ferrofluids, thereby enhancing recoveries of rarelyoccurring biological entities. The ideal aggregating factor is one whichmediates a reversible aggregating effect. Reversal of aggregationeliminates magnetic nanoparticle clusters so as to facilitatevisualization of isolated cells. The factors of the invention operate ina manner similar to the ideal magnetic particle conditions describedabove i.e., by converting colloid nanoparticles which are bound totarget into large particles with the added ability to readily reversethat process.

[0059] The identification and elucidation of an endogenous factorpresent in the blood of most normal donors which enhances the efficiencyof isolation of low density receptor rare cells is described herein.

[0060] Preferable FFAFs include specific multivalent substances whichrecognize determinant(s) on ferrofluid magnetic particles therebycrosslinking the particles. This factor may naturally occur in plasma ormay be an exogenously added reagent. Several types of exogenous reagentsare suitable for this purpose and include, but are not limited to, IgG,dimeric IgG, IgM, Streptavidin, Avidin, Protein A, Protein G, dimeric ortetrameric poly-A or poly-T, or specific oligonucleotide sequences. Thesecondary ligand can be introduced onto ferrofluid and is recognized byFFAF. There are several types of secondary ligands which may be selectedsuch as hapten, biotin, biotin analogues (iminobiotin, desthiobiotin),sheep IgG, goat IgG, rat IgG, poly-A or poly-T or oligonucleotide.FFAF-secondary ligand interaction may be either reversible orirreversible but a reversible interaction is preferred. There areseveral reagents which may be used to reverse FFAF-secondary ligandinteraction such as reducing agents, excess of haptens, excess of haptenanalogues, excess of analogues of the secondary reagent, change of saltconcentration, change of pH or change of temperature.

[0061] When an assay or separation is performed under optimalconditions, the percent recovery of the target cells is maximized.However, the ability to modify the conditions is apparent when exogenousaggregation is induced. In the case of high density surface receptortarget cells, the amount of ferrofluid needed for maximum recovery isquite high without exogenous aggregation, to ensure that each target ismagnetically responsive by saturating all the available binding sites.One benefit of this invention is that less total ferrofluid is neededfor separation, as it does not need to saturate all the availablebinding sites. This is due to the ability to form crosslinks of magneticparticles, mediated by the exogenous aggregation factor and increase themagnetic mass per binding site. Instead of rendering cells magneticallyresponsive by the use of many single magnetic particles per cell, i.e.one particle binds one cell surface antigen, the aggregates of multipleparticles can bind the single antigen and will maintain the samemagnetic force as optimally captured cells. Each particle now has thecapability to bind a cell surface antigen or another particle. In otherwords, even though less total ferrofluid is used, each target cell willhave the same magnetic responsiveness, and the resulting separationefficiency will be comparable to that obtained under optimal conditions.Optimally, 10 μg of ferrofluid is used per 1 ml of sample. Thisconcentration may be reduced 10-fold in accordance with the presentinvention.

[0062] In addition to reducing the amount of ferrofluid needed, themagnetic strength as well as the incubation time can be reduced. Becausethe aggregates of the invention are larger than individual magneticparticles in a non-aggregating separation, a lesser magnetic fieldstrength can still move the aggregates. Promoting exogenous aggregationcreates temporary and reversible large magnetic particles from smallmagnetic particles. Indeed, the benefits of large particles, includingthe ability to use weak magnetic fields for separation, can be appliedto the present invention. The quadrupole magnetic separators typicallyused maintain a field gradient strength of 6.3 kGauss/cm at the vesselsurface. Magnetic arrangements, such as dipoles, which have almost halfthe magnetic field strength at the vessel surface may be utilized inpracticing the methods of the present invention.

[0063] In the non-aggregating system, longer incubation times arerequired to increase the number of magnetic particles per cell foreffective separation. In this system, one magnetic particle will bindone cell surface antigen. However, in an exogenously-induced aggregationsystem, the same number of particles per cell can be achieved by causingmultiple particles per antigen, via the induced aggregates. This allowsthe incubation time to be shortened because not all of the availablebinding sites will need to be bound to magnetic particles. By addingthis second binding pair member, the particles now can bind to otherparticles, instead of only being capable of binding to free cell surfaceantigens. Therefore, as explained above, the binding sites do not needto be saturated to have the same magnetic responsiveness as optimalconditions, which allows the reduction of incubation time. The minimumtime for magnetic incubations is 30 minutes. Using the presentinvention, these times may be reduced up to 3-fold. However, it is notintended that both ferrofluid concentration be reduced and incubationtime be shortened simultaneously. Variations of these steps should beexploited independently of one another in order to maintain maximalrecovery of target bioentities.

[0064] A secondary ligand which is recognized by an exogenous ferrofluidaggregation factor will be coupled to ferrofluid in addition to theabove monoclonal antibody by standard coupling chemistry. The secondaryligand may be a small molecule such as hapten or biotin analogue or abig molecule such as an antibody or a specific protein or a polymer suchas polypeptides or polyoligonucleotides. A biotin analogue such asdesthiobiotin is preferred for conjugation to magnetic particles as asecondary ligand in this invention as it exhibits a lower affinity(Ka=10⁶ M⁻¹ for streptavidin, as compared to native biotin (Ka=10¹⁵M⁻¹).

[0065] The interaction between streptavidin and desthiobiotin can beeasily disrupted by the addition of excess biotin. The combination ofdesthiobiotin and avidin has been used to remove magnetic particles orinsoluble phase from the target substances (PCT/US94/10124 and U.S. Pat.No. 5,332,679). In this invention, that combination is used only toaggregate and disaggregate magnetic particles and not to remove magneticparticles from the target substance.

[0066] The reaction vessel for use in this invention may be either glassor plastic, however, plastic tubes are preferred. The bottom of the tubemay be round or conical in shape. Tubes with different lengths ordiameters may be used to process different volumes of samples. Forexample, in some instances a 50 ml conical tube may be used to process20 ml of blood or more. In one embodiment of this invention, a 12×75 mmpolystyrene tube or 15 ml conical tube is used. The reaction vessel usedduring incubation with magnetic particles and the vessel used duringmagnetic separation does not necessarily need to be the same. Twodifferent types of vessels may be used, one type for incubation andanother type for magnetic separation. However, it is preferred to useonly one vessel in both cases. The magnetic separation vessel may be atube or a flow-through chamber or some other device.

[0067] The test medium used in practicing the present invention may beany liquid or solution which contains the target substance and ispreferably blood. A test sample in the reaction vessel is incubated witha ferrofluid conjugated to antibodies specific for a target substanceand a secondary ligand specific for FFAF. Additionally, an exogenousFFAF is added simultaneously with ferrofluid to the test sample or afterbinding of ferrofluid to target substance. Optionally, a reagent whichinhibits or disables naturally occurring aggregating factor may be addedsimultaneously with ferrofluid, or added prior to ferrofluid addition.After an optimum incubation time, magnetically labeled targets areseparated from the rest of the test medium in a magnetic separator. Themagnetic separator and separation time are selected based upon testmedium and reaction vessel. It is preferred to use high-gradientmagnetic separation devices such as those described in U.S. Pat. No.5,186,827. After aspirating the uncollected liquid, the collected cellsmay be resuspended in an isotonic buffer or permeabilizing solution topermeabilize cells for intracellular staining. The magnetically labeledcells are reseparated magnetically to remove permeabilizing reagents.The collected cells are resuspended in a small volume of cell buffer forstaining with labeling substances. The volume of the buffer may be from100-300 μl. Optionally the cell buffer may contain staining antibodies.Additionally, the cell buffer may contain a disaggregating reagent asdescribed above, e.g., biotin. The final concentration of biotin may befrom 1-5 mM. The incubation time for antibody staining or for ferrofluiddisaggregation with disaggregating reagent may be from 10-60 minutes andis preferably 15 minutes. After optimum staining with antibodies ordisaggregation of ferrofluid, excess reagents may be removed from cellsby another magnetic separation. After aspirating the uncollected liquid,the collected cells are resuspended in a small volume of isotonicbuffer. The volume of this buffer may be from 100-500 μl. The ferrofluidlabeled cells may be further processed or analyzed by flowcytometry ormicroscopy.

[0068] While magnetic particles conjugated to antibody only have beendescribed above, other types of conjugated magnetic particles arecontemplated for use in the present invention. Magnetic particlesconjugated to proteins other than antibodies may be used. For example,streptavidin conjugated magnetic particles may be used to bind targetcells which are labeled with antibody-biotin conjugates. Followinglabeling of target cells, excess unbound antibody-biotin may be removedby a wash step using a centrifuge. The target cells labeled withantibody-biotin are then incubated with streptavidin ferrofluid formagnetic labeling of cells. Desthiobiotin conjugated to any polymer orprotein (aggregating factor) will be added to the test medium toaggregate ferrofluid. Aggregating factor may be added simultaneouslywith magnetic particles or after the magnetic particles bind the targetcells. The number of desthiobiotins per polymer or protein should bemore than one to aggregate ferrofluid. Preferably desthiobiotinconjugated to bovine serum albumin (BSA) may be used. The number ofdesthiobiotins on BSA may be 2-10. Such desthiobiotin/protein conjugatesmay be synthesized as set forth hereinbelow.

[0069] Although the present invention is described herein primarily withreference to tumor cell selection from blood, the invention is notlimited to tumor cell selection. Other cell types present in blood,leukophoresis or bone marrow, such as CD34, CD4, and fetal cells may beselected. The antigenic determinants on those cells may be low to high.More generally, the invention applies to the isolation of any cell whichrequires magnetic enhancement for its efficient isolation.

[0070] The following examples are provided to illustrate variousembodiments of the invention. These examples are not intended to limitthe scope of the invention in any way.

EXAMPLE I

[0071] The following data illustrate the effects of the FFAF of thepresent invention on the recovery of low and high density receptor tumorcells spiked into blood samples. An exemplary FFAF has been identifiedas a specific IgM present in the blood samples of most donors. Reducingagents such as dithiothreitol (DTT) and mercaptoethane sulfonic acid(MES) which cleave disulfide linkages, prevented ferrofluid aggregationin blood by converting pentameric IgM to its monomeric form. DTT is nota preferred reagent for use in the methods of the present invention, ashigh concentrations alter cellular morphology, and are toxic to targetcells and leukocytes.

[0072] In this example, the effect of MES on ferrofluid aggregation andtumor cell recovery of both high and low antigen density tumor cellsfrom spiked blood is described. The protocol used for this study was asfollows. Blood (2 ml) was placed in a 12×75 mm polystyrene tube and 1 mlof Immunicon dilution-wash buffer was added to dilute the blood. Next,100 μl of cell buffer (isotonic 7 mM phosphate, pH 7.4 with 1% BSA and50 mM EDTA) containing approximately 1000 SKBR3 or PC3 cells was added.Increasing volumes of MES (not exceeding 150 μl) were added to the bloodsamples to obtain different concentrations of reducing agent. Aftermixing, EpCAM MAb (GA73.3; 50 μl) conjugated ferrofluid magneticparticles were added to the sample. The final concentration of magneticparticles was 5 μg/ml. The blood sample was mixed well and incubated for15 minutes at room temperature. After the incubation, the tubecontaining the blood sample was placed in a quadrupole magneticseparation device. Magnetic separation was performed for 10 minutes. Thesupernatant was aspirated and the tube was removed from the magneticdevice. The magnetically collected cells were resuspended in 1 ml ofdilution-wash buffer and reseparated in a quadrupole magnetic separationdevice for 5 minutes. The supernatant was discarded and after removalfrom the quadrupole device, the target cells were resuspended in 150 μlof dilution wash buffer. A portion of this sample (5 μl) was spotted ona microscope slide. The recovered cells were then photographed using amicroscope with a digital camera attached to it.

[0073] The remaining sample was subjected to flowcytometry analysis toassess the recovery of tumor cells using the following procedure.Phycoerythrin (PE)-conjugated MAb (5 μl) specific for tumor cells (Neu24.7) and 5 μl of peridinin chlorophyll protein (PerCP)-conjugated CD45monoclonal antibody were added to the sample which was then incubatedfor 15 minutes. After the incubation, 1 ml of dilution-wash buffer wasadded and a magnetic separation was performed in order to remove excessstaining antibodies. The magnetically collected cells were resuspendedin 500 μl of dilution-wash buffer. Nucleic acid dye (10 μl) and 5 μl of3 mM sized fluorescent beads (5000) were added to this sample. Thesample was then analyzed on a FACSCalibur flowcytometer (BectonDickinson) using FL1 as threshold. The fraction of the fluorescent beadsacquired in the flowcytometer was used to determine the amount of sampleanalyzed by flowcytometry which, in turn, facilitates calculation of therecovery of spiked tumor cells. Recovery of tumor cells (%)Concentration of MES (mM) SKBR3 PC3  0 77 46 20 80 50 50 80 31 75 82 27100 70 17

[0074] When viewed by microscopy, the final sample showed freeferrofluid aggregates and ferrofluid aggregates on tumor cells in theabsence of MES. As the concentration of MES was increased, ferrofluidaggregates decreased and no aggregates were seen at higherconcentrations of MES. These visual results were then compared withtumor cell recovery as measured by flowcytometry. The addition of MEShad no significant effect on recovery of SKBR3 cells (high antigendensity) although microscopy revealed that it decreased ferrofluidaggregation in solution and on cell surfaces. In contrast, MES had asignificant effect on the recovery of PC3 cells (low antigen density).As the concentration of MES was increased from 0-100 mM, recovery wasdecreased from 47% to 17%. This decrease in recovery of PC3 cells in thepresence of increasing concentrations of MES was due to inhibition offerrofluid aggregation and not due to any side effects of MES on cells,as MES did not decrease the recovery of same spiked PC3 cells from lysedblood samples. Lysed blood is obtained by lysing red blood cells withammonium chloride followed by wash step which removes plasma andammonium chloride. Lysed blood samples contain only leukocytes (whitecells) whereas whole blood also contains erthyrocytes and plasma. Noferrofluid aggregation is observed with lysed blood. Moreover, MES hasno significant effect on cell morphology. These data show that lowantigen density cells were isolated less efficiently than high antigendensity cells and inhibition of ferrofluid aggregation dramaticallyaffects the isolation of low antigen density cells. Ferrofluidaggregation was also not observed when washed blood (blood cells withplasma removed) samples were utilized Therefore, washed blood sampleswere used as a control for no aggregation. Just as with whole blood, therecovery of SKBR3 cells was not decreased with washed blood. PC3 cellrecovery on the other hand was decreased significantly (2 to 5-fold)when washed blood was used. This data clearly shows that ferrofluidaggregation does not have any effect on SKBR3 cells recovery but has amajor effect on PC3 cells recovery.

[0075] In summary, aggregation of tumor specific ferrofluid with theplasma component (IgM) present in blood of many patients at varyinglevels has a significant effect on recovery of low antigen densitycells. Recovery of such cells is affected by the extent of ferrofluidaggregation and increases with increasing aggregation. Ferrofluidaggregation increases the recovery of low antigen density cells byincreasing magnetic load on the cells. Ferrofluid aggregation can varyfrom one blood donor to another depending upon the concentration of theaggregating factor or aggregator. As a result, the recovery of tumorcells will vary from person to person even though they may possess thesame number of circulating tumor cells. It is also possible that theconcentration of the aggregator present in the blood from the sameperson can vary with time thus altering the extent of ferrofluidaggregation and recovery of tumor cells. The best way to prevent thisvariation is to prevent naturally occurring ferrofluid aggregation.However, this gives rise to a decrease in the efficiency of tumor cellsisolation and detection. One means to increase tumor cell recovery underthese circumstances will be to improve the magnetic device with a highergradient which can pull weakly magnetic labeled cells effectively andincrease their recovery. The other way to increase the recovery of tumorcells will be to mimic natural ferrofluid aggregation with an exogenousreagent. This reagent can be a specific multivalent reagent which canrecognize ferrofluid and can be added to the blood and ferrofluid. Thespecific reagent will aggregate ferrofluid similarly to IgM but under acontrolled reaction. Controlled aggregation will have two advantages:(1) the percentage of tumor cells recovered will increase; and (2) thepercentage of tumor cells recovered will not vary from patient topatient and will not vary with time from the same patient when thesamples have the same number of tumor cells.

EXAMPLE II

[0076] Preparation of Desthiobiotin/EpCAM MAb Ferrofluid for ControlledAggregation.

[0077] A base ferrofluid was made as described in U.S. Pat. No.5,698,271. Monoclonal antibody to the epithelial cell adhesion molecule(EPCAM) was conjugated to base material by standard coupling chemistry,as used in U.S. patent application Ser. No. 09/248,388. EpCAM MAbferrofluid was then resuspended in 20 mM HEPES, pH 7.5 for conjugationto desthiobiotin using N-hydroxysuccinimide-DL-desthiobiotin(NHS-desthiobiotin) (Sigma, Cat.# H-2134). A stock solution ofNHS-desthiobiotin was made in DMSO at 1 mg/ml. NHS-desthiobiotin (19 μg)was added to 1 mg of EpCAM MAb ferrofluid and incubated at roomtemperature for 2 hours. Unreacted NHS-desthiobiotin was removed bywashing 3 times with 20 mM HEPES, pH 7.5 containing 1 mg/ml BSA, 0.05%ProClin 300 using a high gradient magnet. After the final wash,desthiobiotin/EpCAM MAb ferrofluid was resuspended in Immuniconferrofluid storage buffer and filtered through a 0.2 μm syringe filter.

EXAMPLE III

[0078] Increase of Recovery of Low Antigen Density PC3 Tumor Cells FromSpiked Blood by Aggregation of Desthiobiotin/EpCAM Ferrofluid withStreptavidin.

[0079] In this example, prostate carcinoma cells (PC3) which have a lowEpCAM antigen density were spiked into normal blood and used as a modelsystem to assess recovery of those spiked cells. A known number of PC3cells (˜5000) in 50 μl of buffer (isotonic 7 mM phosphate, pH 7.4 with1% BSA and 50 mM EDTA) were spiked into 1 ml of normal blood withoutplasma in a 12×75 mm polystyrene tube. Blood without plasma was used inthese experiments to prevent any interference of plasma components inthe selection of target cells and it was prepared by centrifugation ofblood. 500 μl of Immunicon dilution-wash buffer and 15 μl ofstreptavidin at different concentrations in PBS were added to aliquotsof the blood sample. After mixing the sample, desthiobiotin/EpCAM MAbferrofluid (25 μl) from Example 1 was added to the sample, mixed welland incubated at room temperature for 15 minutes. The finalconcentration of ferrofluid was 5 μg/ml. After the incubation, the tubecontaining the blood sample was placed in a quadrupole magneticseparator for 10 minutes for collection of magnetically labeled cells.The uncollected sample was aspirated and the tube was removed from themagnetic separator. The magnetically collected cells were resuspended in750 μl of dilution-wash buffer and reseparated in a magnetic separatorfor 5 minutes. The uncollected sample was discarded again and thecollected cells were resuspended in 150 μl of dilution-wash buffer afterremoval of the tube from the magnetic separator.

[0080] The sample was then stained with antibodies to determine therecovery of tumor cells by flowcytometry as follows. 5 μl ofphycoerythrin (PE)-conjugated MAb specific for tumor cells (Neu 24.7)and 5 μl of peridinin chlorophyll protein (PerCP)—conjugated CD45 MAbwere added-to the sample and incubated for 15 minutes. After theincubation, 1 ml of dilution-wash buffer was added and a magneticseparation was performed for 5 minutes in order to remove excessstaining antibodies. The magnetically collected cells were resuspendedin 500 μl of dilution-wash buffer. Nucleic acid dye (10 μl) and 5 μl of3μM fluorescent beads (5000) were added to this sample. The sample wasthen analyzed on a FACSCalibur flowcytometer (Becton Dickinson) usingFL1 as threshold. The fraction of the fluorescent beads acquired in theflowcytometer was used to determine the amount of sample analyzed byflowcytometry which was then used to calculate the recovery of spikedtumor cells. The percent recovery of tumor cells are shown in thefollowing Table. Concentration of Aggregator, Tumor Cells (PC3)Streptavidin (μg/ml) % recovery 0.0 14 0.2 60 0.5 74 2.0 80 5.0 75

[0081] The samples which were left after the flowcytometry analysis weredivided into two parts. Biotin from a stock solution in PBS was added toone part of the sample to final concentration of 2 mM and incubated atroom temperature for 15 minutes to disaggregate streptavidin-mediatedferrofluid aggregates. These samples (5 μl) were spotted on a microscopeslide and photographs of the recovered cells were taken using amicroscope with a digital camera attached to it.

[0082] The data indicate that the recovery of tumor cells (PC3) wasincreased significantly as the concentration of aggregator(streptavidin) was increased, reaching a maximum at a 2 μg/mlconcentration of streptavidin. These results were correlated to freeferrofluid aggregates in solution and ferrofluid aggregates on cells asobserved with microscopy. There were no ferrofluid aggregates at 0 μg/mlof streptavidin and ferrofluid aggregates were increased as theconcentration of streptavidin was increased. Streptavidin causesaggregation of ferrofluid by multivalent binding of streptavidin todesthiobiotin on different ferrofluid particles. All these ferrofluidaggregates were reversibly disaggregated by the addition of excessbiotin. The principle of this disaggregation of ferrofluid by excessbiotin was due to displacement of desthiobiotin from streptavidin asbiotin has a higher affinity than desthiobiotin for streptavidin.

EXAMPLE IV

[0083] Recovery of Spiked Low and High EPCAM Antigen Density Cells fromBlood With and Without Aggregation of Desthiobiotin/EpCAM MAbFerrofluid.

[0084] Breast carcinoma cells (SKBR3) have about 7-times higher EPCAMantigen density compared to PC3 cells and were chosen as the model highantigen density tumor cells for this example. A known number of SKBR3 orPC3 cells in cell buffer were spiked into 1 ml of blood without plasmaseparately in a 12×75 mm tube. Ferrofluid dilution-wash buffer (500 μl)and 15 μl of PBS containing streptavidin were added to the sample. Aftermixing the sample, 25 μl of desthiobiotin/EpCAM MAb ferrofluid was addedand the blood sample mixed well and incubated for 15 minutes. Afterincubation, the tube was placed in a quadrupole magnetic separator for10 minutes to collect magnetically labeled cells. The magneticallyisolated cells were analyzed for recovery of tumor cells byflowcytometry and for observation of cells by microscopy as described inExample II. Concentration of aggregator, PC3 cells SKBR3 cellsStreptavidin recovery recovery (μg/ml) (%) (%) 0 23 91 2 77 98

[0085] The data reveal a significant difference in recovery of tumorcells between low and high antigen density cells when the ferrofluidaggregator, streptavidin, was not added to the blood sample. There werealso no ferrofluid aggregates in solution or on cell surfaces withoutstreptavidin as observed with microscopy. Addition of streptavidin tothe blood sample increased the recovery of low antigen density PC3 cellssignificantly (3-fold) with a commensurate increase of ferrofluidaggregation in solution and on the cells. On the other hand, there wasonly a small difference in recovery of high antigen density SKBR3 cellswith and without streptavidin present in the blood sample. There wereenough ferrofluid particles on SKBR3 cells even without ferrofluidaggregation to collect them effectively and to recover all of them.Ferrofluid aggregates in solution and on cells were completelydisaggregated by the addition of excess biotin to the sample. In thecase of low antigen density cells, there were not enough ferrofluidparticles on cells to be collected effectively by magnetic methods.Ferrofluid aggregation by streptavidin increased the number of particleson these cells facilitating collection, effectively resulting higherrecovery. It is also noteworthy that aggregation of ferrofluid increasedthe recovery of low antigen density cells close to that obtained withthe high antigen density cells. In other words, there was no significantdifference in recovery of low and high antigen density tumor cells uponaddition of reversible ferrofluid aggregator to the blood sample.

EXAMPLE V

[0086] Inhibition of Ferrofluid Aggregation by Endogenous AggregationFactors and Creation of Controlled Ferrofluid Aggregation with anExogenous Aggregation Factor.

[0087] In this example, a method is provided to inhibit all endogenousferrofluid aggregation factors and to create controlled ferrofluidaggregation by addition of an exogenous aggregation factor. Theendogenous ferrofluid aggregation factors present in the sample can beinhibited by adding a variety of inhibitors to the sample. Theseinhibitors will act on different endogenous aggregation factors andprevent them from either crosslinking or binding to ferrofluid to causeaggregation. Inhibition will eliminate any variations in ferrofluidaggregation from sample to sample as endogenous aggregation factors arepresent at different concentrations in different samples. Onceendogenous factor-ferrofluid aggregation is prevented, ferrofluidaggregation can be promoted by adding an exogenous aggregation factorwhich can enhance the recovery of targets efficiently. The exogenousaggregation can be controlled consistently with all the samples and itcan be readily reversed.

[0088] The blood sample is preincubated with a buffer containinginhibitors to inhibit endogenous ferrofluid aggregation factors beforeferrofluid is added to the blood. The antibody coupled ferrofluidcontains bovine serum albumin and streptavidin on the surface offerrofluid particles in addition to antibody. Therefore, the possibleferrofluid aggregation factors can be IgM (specific for crystalsurface), human-anti-mouse antibody (HAMA), human-anti-bovine serumalbumin antibody (HABAA), human-anti-streptavidin etc. If any of theabove aggregation factors are present in plasma, they will recognize andbind to ferrofluid and cause ferrofluid to aggregate. It is alreadyknown that some patient plasma samples have HAMA and HABAA presenttherein. Clearly, any other components used to manufacture ferrofluidscould also be targets for aggregation and would needs to be dealt withaccordingly.

[0089] One of the inhibitors can be a reducing agent, such asmercaptoethane sulfonic acid at 100 mM, which can disable IgM-inducedaggregation without affecting the ligands used for labeling cells. Thereducing agent can be added as a single reagent to the blood or could beplaced in a blood collection tube. The second inhibitor can be bovineserum albumin, which can be included in the buffer at 10 mg/ml, and willneutralize any HABAA. The third inhibitor can be nonspecific mouseantibody, in particular, the appropriate isotype which matches theantibody on the ferrofluid. This can be included in the buffer at aconcentration of 0.5-5 mg/ml to neutralize even the most severe HAMA.The fourth inhibitor can be Streptavidin to be included in the buffer,if necessary, to neutralize any anti-streptavidin antibody present inplasma. However, there is no any information regarding the existence ofanti-streptavidin antibody in plasma at this date.

[0090] The pre-treatment of blood with the above buffer and reducingagent can be from 15-30 minutes to neutralize all endogenous aggregationfactors. After all endogenous aggregation factors are neutralized, anexogenous ferrofluid aggregation factor is added to the sample, followedby ferrofluid. The ferrofluid is coupled to an antibody specific fortargets, as well as to another ligand specific for the exogenousaggregation factor. After optimum labeling of target cells withferrofluid and induced aggregation of ferrofluid with exogenousaggregation factor, the sample is subjected to magnetic separation toenrich targets. After removing all non-targets, magnetically-labeledtargets and free ferrofluid are resuspended in a small volume of buffer.The magnetically-labeled targets, such as cells, can be permeabilized tostain intracellular antigens. The sample is then incubated withdifferent staining reagents depending upon the desired analysis method,including flow cytometry or fluorescent or bright field microscopy.After optimum incubation time, the excess staining reagents are removedby wash step using magnetic separation. The magnetically-labeled cellsare then resuspended in a small volume of buffer. The final samplecontains free ferrofluid aggregates and aggregates on target cells. Thefinal sample without any further treatment can be used for flowcytometryanalysis, as ferrofluid aggregation on cell surface does not interferewith the analysis. However, ferrofluid aggregation on cell surfaceinterferes with microscopy analysis.

[0091] In such cases, exogenous mediated-ferrofluid aggregation shouldbe reversed. This can be achieved by resuspending the final sample in abuffer containing a disaggregation factor which binds to exogenousaggregation factor. The disaggregation factor disaggregates allferrofluid aggregates, leaving cells easy to view and analyze. Thesemethods permit effective target recovery and visualization formorphology studies.

[0092] Several patents and pending U.S. patent applications are referredto in the present specification. The entire disclosures of each of thesepatents and patent applications are incorporated by reference herein.

[0093] While certain of the preferred embodiments of the presentinvention have been described and specifically exemplified above, it isnot intended that the invention be limited to such embodiments. Variousmodifications may be made thereto without departing from the scope andspirit of the invention, as set forth in the claims.

What is claimed is:
 1. A method for isolating a target bioentity from abiological sample by means of colloidal magnetic particles with reducedaggregation of said magnetic particles, comprising: a) obtaining abiological specimen suspected of containing said target bioentitytogether with non-target bioentities and endogenous aggregating factors;b) contacting said biological specimen with a reagent effective toinactivate any endogenous aggregating factors present in said specimen;c) preparing an immunomagnetic suspension comprising a mixture of saidspecimen and colloidal, magnetic particles coupled to a biospecificligand having binding affinity for at least one characteristicdeterminant present on said target bioentity; and d) subjecting saidimmunomagnetic suspension to a magnetic field to obtain a targetbioentity- enriched fraction.
 2. A method as claimed in claim 1, furthercomprising the steps of e) purifying said target bioentity from saidenriched fraction; and f) analyzing said purified target bioentity.
 3. Amethod as claimed in claim 1, further comprising the steps of: e) addingto said target bioentity enriched fraction at least one biospecificreagent which has binding affinity for at least one additionalcharacteristic determinant on said target bioentity; f) separating saidtarget bioentity in a magnetic field to remove unbound biospecificreagent from said enriched fraction.
 4. A method as claimed in claim 3,further comprising the steps of: g) purifying said separated targetbioentity; and h) analyzing said purified target bioentity.
 5. A methodas claimed in claim 3, further comprising the steps of: g) adding anon-cell exclusion agent to said separated target bioentities to allowexclusion of non-nucleated entities present in the sample; h) purifyingsaid target bioentity; and i) analyzing said purified targetbioentities.
 6. A method as claimed in claim 1, wherein said targetbioentity is selected from the group consisting of tumor cells, virallyinfected cells, fetal cells in maternal circulation, virus particles,bacterial cells, white blood cells, myocardial cells, epithelial cells,endothelial cells, proteins, hormones, DNA, and RNA.
 7. A method asclaimed in claim 2, wherein said target bioentities are analyzed by aprocess selected from the group consisting of multiparameter flowcytometry, immunofluorescent microscopy, laser scanning cytometry,bright field base image analysis, capillary volumetry, manual cellanalysis and automated cell analysis.
 8. A method as claimed in claim 1,said aggregation inhibiting agent being at least one selected from thoseconsisting of a reducing agent, an animal serum protein, animmune-complex, a carbohydrate, a chelating agent, an unconjugatedferrofluid, and diamino butane.
 9. A method as claimed in claim 5,wherein said aggregation inhibiting agent is a reducing agent selectedfrom the group consisting of mercapto ethane sulfonic acid [MES],mercapto propane sulfonic acid [MPS] and dithiothreitol [DTT].
 10. Amethod as claimed in claim 1, wherein said biospecific ligand is amonoclonal antibody.
 11. A method as claimed in claim 9, wherein saidbiospecific ligand is a monoclonal antibody having affinity forepithelial cell adhesion molecule.
 12. A method for isolating targetbioentities from a biological sample by means of colloidal magneticparticles with controlled aggregation of said magnetic particles,comprising: a) obtaining a biological specimen suspected of containingsaid target bioentities together with non-target bioentities andendogenous aggregating factors; b) contacting said biological specimenwith a reagent effective to inactivate any endogenous aggregatingfactors present in said specimen; c) preparing an immunomagneticsuspension comprising a mixture of colloidal, magnetic particles coupledto a biospecific ligand having affinity for at least one characteristicdeterminant present on said target bioentity, said magnetic particlesbeing further coupled to a first exogenous aggregation enhancing factorwhich comprises one member of a specific binding pair; d) adding asecond exogenous aggregation enhancing factor to said immunomagneticsuspension to increase aggregation of said particles, said secondaggregating enhancing factor comprising the other member of saidspecific binding pair; and e) subjecting said sample to a magnetic fieldto obtain a target bioentity-enriched fraction.
 13. A method as claimedin claim 12, further comprising the steps of: f) adding to saidimmunomagnetic suspension at least one biospecific reagent havingbinding affinity for at least one additional characteristic determinanton said target bioentity; g) separating said target bioentities in amagnetic field to remove unbound biospecific reagent; and h) adding anon-cell exclusion agent to said separated bioentities to allowexclusion of non-nucleated entities present in the sample; i) purifyingsaid target bioentities; and j) analyzing said separated and purifiedtarget bioentities.
 14. A method as claimed in claim 12, furthercomprising examining said purified target bioentity-enriched fraction todetermine the degree of aggregation mediated by said first and secondmembers of said specific binding pair.
 15. A method as claimed in claim12, wherein one or the other member of said specific binding pair isadded to said purified bioentity fraction to reverse aggregation of saidsample, thereby facilitating analysis of said target bioentities.
 16. Amethod as claimed in claim 12, wherein said specific binding pair isselected from the group consisting of biotin-streptavidin,antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist,lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotinanalog-streptavidin, biotin analog-avidin, desthiobiotin-streptavidin,desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.17. A method as claimed in claim 12, wherein said biospecific ligand isa monoclonal antibody.
 18. A method as claimed in claim 17, wherein saidbiospecific ligand is an antibody having affinity for epithelial celladhesion molecule.
 19. A method as claimed in claim 12 wherein said atleast one biospecific reagent is selected from the group of consistingof monoclonal antibodies, polyclonal antibodies, detectably labeledantibodies, antibody fragments, and single chain antibodies.
 20. Amethod as claimed in claim 12 wherein said target bioentities areanalyzed by a process selected from the group consisting ofmultiparameter flow cytometry, immunofluorescent microscopy, laserscanning cytometry, bright field base image analysis, capillaryvolumetry, manual cell analysis and automated cell analysis.
 21. Amethod as claimed in claim 12, wherein said immunomagnetic suspension isincubated for less than 30 minutes.
 22. A method as claimed in claim 12,wherein said purification of said sample is performed in a magneticfield gradient of less than 6.3 kGauss/cm.
 23. A method as claimed inclaim 12, wherein said colloidal magnetic particle concentration in saidimmunomagnetic suspension is less than 10 μg per milliliter.
 24. Amethod for isolating low antigen density tumor cells from a biologicalsample by means of colloidal magnetic particles with controlledaggregation of said magnetic particles, comprising: a) obtaining abiological specimen suspected of containing said tumor cells togetherwith non-tumor cells and endogenous aggregating factors; b) preparing animmunomagnetic suspension comprising a mixture of said specimen andcolloidal, magnetic particles coupled to a biospecific ligand havingbinding affinity for at least one characteristic determinant present onsaid tumor cell, said magnetic particles being further coupled to afirst exogenous aggregation enhancing factor, said factor comprising onemember of a specific binding pair; c) adding a second exogenousaggregating enhancing factor to said immunomagnetic suspension toincrease aggregation of said particles, said second aggregatingenhancing factor comprising the other member of said specific bindingpair; and d) purifying said sample in a magnetic field to obtain a tumorcell-enriched fraction.
 25. A method as claimed in claim 24, furthercomprising the steps of: e) adding to said fraction, at least onebiospecific reagent having binding affinity for at least one additionalcharacteristic determinant on said tumor cell; f) separating said tumorcells in a magnetic field to remove unbound biospecific reagent; g)adding a non-cell exclusion agent to said separated cells to allowexclusion of non-nucleated entities present in the sample; and h)analyzing said separated tumor cells to assess at least one of tumorcell number and type.
 26. A method as claimed in claim 25, wherein amember of said specific binding pair is added to the separated cells toreverse aggregation of said sample, thereby facilitating analysis ofsaid cells.
 27. A method as claimed in claim 24, wherein said specificbinding pair is selected from the group consisting ofbiotin-streptavidin, antigen-antibody, receptor-hormone,receptor-ligand, agonist-antagonist, lectin-carbohydrate, ProteinA-antibody Fc, and avidin-biotin, biotin analog-avidin,desthiobiotin-streptavidin, desthiobiotin-avidin,iminobiotin-streptavidin, and iminobiotin-avidin.
 28. A method asclaimed in claim 24, wherein said biospecific ligand is a monoclonalantibody.
 29. A method as claimed in claim 28, wherein said biospecificligand is an antibody having binding affinity for epithelial celladhesion molecule.
 30. A method as claimed in claim 25, wherein said atleast one biospecific reagent is selected from the group of consistingof monoclonal antibodies, polyclonal antibodies, detectably labeledantibodies, antibody fragments and single chain antibodies.
 31. A methodas claimed in claim 25 wherein said tumor cells are analyzed by aprocess selected from the group consisting of multiparameter flowcytometry, immunofluorescent microscopy, laser scanning cytometry,bright field base image analysis, capillary volumetry, manual cellanalysis and automated cell analysis.
 32. A method as claimed in claim24 wherein said biological sample is pretreated with an aggregationinhibiting agent to inactivate endogenous aggregation factors present inthe sample prior to the preparation of said immunomagnetic suspension.33. A method as claimed in claim 25, wherein said purified tumorcell-enriched fraction is examined to determine the degree ofaggregation mediated by said first and second members of said specificbinding pair.
 34. A kit for inhibiting endogenous aggregation ofcolloidal magnetic particles in processing of biological material forisolation of target bioentities from such materials, comprising: a)coated magnetic nanoparticles comprising a magnetic core material, aprotein base coating material, and an antibody that binds specificallyto a first characteristic determinant of said target bioentity, saidantibody being coupled, directly or indirectly, to said base coatingmaterial; b) at least one antibody having binding specificity for asecond characteristic determinant of said target bioentity; c) anendogenous aggregation inhibiting factor; and d) a non-cell exclusionagent for excluding non-nucleated cells from analysis.
 35. A kit forisolating low antigen density tumor cells from a biological sample withcontrolled aggregation of colloidal magnetic particles, comprising: a) areagent effective to inactivate endogenous aggregating factors; b)coated magnetic nanoparticles comprising a magnetic core material, aprotein base coating material, and an antibody that binds specificallyto a first characteristic determinant of said tumor cell, said antibodybeing coupled, directly or indirectly, to said base coating material;said magnetic particles being further coupled to a first exogenousaggregation enhancing factor, said factor comprising one member of aspecific binding pair; c) at least one antibody having bindingspecificity for a second characteristic determinant of said tumor cell;d) a second exogenous aggregation enhancing factor, said secondaggregation factor comprising the other member of said specific bindingpair; and e) a non-cell excluding agent for excluding non-nucleatedcells from analysis.
 36. A kit as claimed in claim 35, wherein saidspecific binding pair is selected from the group consisting ofbiotin-streptavidin, antigen-antibody, receptor-hormone,receptor-ligand, agonist-antagonist, lectin-carbohydrate, ProteinA-antibody Fc, and avidin-biotin, biotin analog-avidin,desthiobiotin-streptavidin, desthiobiotin-avidin,iminobiotin-streptavidin, and iminobiotin-avidin.
 37. A kit as claimedin claim 35, said kit further comprising a reagent for reversing theeffect of said exogenous aggregation factor.
 38. A kit as claimed inclaim 35, said reagent effective to inhibit endogenous aggregationfactor being at least one selected from the group consisting of areducing agent, an animal serum protein, an immune-complexes, acarbohydrate, a chelating agent, an unconjugated ferrofluid, and diaminobutane.
 39. A method as claimed in claim 38, wherein said saidendogenous aggregation factor inhibiting reagent is a reducing agentselected from the group of agents consisting of mercapto ethane sulfonicacid [MES], mercapto Propane Sulfonic acid [MPS] and dithiothreitol[DTT].